DNA coding for a human vasoconstrictive peptide and use thereof

ABSTRACT

Disclosed are (1) a DNA containing a cDNA segment coding for human endothelin-2 (SEQ ID NO:1), (2) a precursor of human endothelin-2 (SEQ ID NO:2), (3) a transformant carrying a DNA containing a cDNA segment coding for human endothelin-2, and (4) a method for preparing mature human endothelin-2 which comprises culturing the transformant described in (3), accumulating a protein in a culture medium, and collecting the same, whereby human endothelin-2 and the precursors thereof can be produced in large amounts.

FIELD OF THE INVENTION

The present invention relates to a DNA containing a cDNA segment codingfor a human vasoconstrictive peptide (SEQ ID NO:1), namely humanendothein-2, a precursor protein of human endothelin-2 (SEQ ID NO:2) anda method for preparing endothelin-2.

In this specification, the term “precursor protein” is preferably usedto describe a protein which includes an amino acid sequence of a maturepeptide and has a portion or all of an amino acid sequence coded with aDNA segment of the peptide at the N-terminus, the C-terminus or bothtermini thereof.

BACKGROUND OF THE INVENTION

There have been reports of endothelium-dependent vasoconstrictorreactions to various mechanical and chemical stimuli as well asendothelium-dependent vasodilative reactions. For example, it is knownthat vasoconstriction can be induced by mechanical loads such asvascular stretch and increased vascular inner pressure, by such agentsas thrombin and by hypoxemia, and further that noradrenaline-inducedvasoconstriction can be enhanced by use of neuropeptide Y [Proc. Natl.Acad. Sci. U.S.A. 79, 5485 (1982); ibid. 81, 4577 (1984)].

Endothelial cell-derived coronary vascular constrictor factors (eachhaving molecular weights of 8,500 and 3,000) are described in Amer. J.Physiol. 248, c550 (1985) and J. Cell Physiol. 132, 263 (1987). However,their sequences are unknown. An endothelial cell-derived peptide-likesubstance is also described in J. Pharmacl. Exp. Ther. 236, 339 (1985).However, the sequence of that substance is also unknown.

On the other hand, vasopressin is known as a peptide having avasoconstrictor activity. The amino acid sequence of vasopression hasbeen determined. There have been no reports, however, that vasopressinwas obtained from mammalian or bird vascular endothelial cells. Althoughthere is a report that an angiotensin having a vasoconstrictor activitywas obtained from the endothelial cells of bovine aortas [CirculationResearch 60, 422 (1987)], the angiotensin is a peptide having amolecular weight of only about 1,000.

Some of the present inventors have previously succeeded in isolatingporcine endothelin as a peptide having a similar vasoconstrictoractivity from the endothelial cells of porcine aortas (Japanese PatentApplication No. 255381/1987). Some of the present inventors have alsosucceeded in isolating human endothelin and cloning porcine endothelincDNA and human endothelin cDNA (Japanese Patent Application Nos.275613/1987, 313155/1987 and 148158/1988). The mature polypeptides ofthe porcine endothelin and the human endothelin have the same amino acidsequence, and are referred to as endothelin-1.

Further, the present inventors have filed patent applications withrespect to the isolation of rat endothelin and the cloning of its cDNA(Japanese Patent Application Nos. 174935/1988 and 188083/1988), and thisis referred to as endothelin-3.

Furthermore, the present inventors have also filed a patent applicationwith respect to the cloning of human endothelin-3 (Japanese PatentApplication No. 278497/1989).

Moreover, the present inventors have also filed a patent applicationwith respect to the isolation of mouse endothelin and the cloning of itscDNA (Japanese Patent Application No. 223389/1988), and this is referredto as endothelin B.

In addition, the present inventors have cloned, from a genomic humanlibrary, a DNA coding for endothelin having an amino acid sequencedifferent from that of endothelin-1 which has been named endothelin-2,and have filed a patent application with respect to a protein ofendothelin-2 and its DNA (Japanese Patent Application No. 274990/1989).

The amino acid sequences of these endothelin-1 (SEQ ID NO:3), endothelinB (SEQ ID NO:4), endothelin-3 (SEQ ID NO:5) and endothelin-2 (SEQ IDNO:6) are shown in FIG. 1 in comparison to one another.

Endothelin is a general term for peptides having a molecular weight of2500±300 and having 21 amino acid residues, including four cysteinegroups located at the 1st, 3rd, 11th and 15th residues from theN-terminus of the amino acid sequence, which form two sets of disulfidebonds. One of the combinations of the disulfide bonds may be 1-15 and3-11 cysteine groups, and the other may be 1-11 and 3-15. The formercombination is higher in ratio of formation and in activity. than thelatter combination.

As described above, homologous endothelin peptides have been discoveredfrom various animals. However, no novel homologous genes have beendiscovered from the same animal species. It is therefore a currentsubject that novel homologous endothelin is further screened, and thestructure and activity of the endothelin are studied, thereby examiningits usefulness, and that the novel peptide is cloned by recombinant DNAtechnology to allow mass production thereof.

SUMMARY OF THE INVENTION

The present inventors have variously studied, considering that importantcontributions will be made to future studies and medical treatments, ifa novel homologous gene having the vasoconstrictor activity describedabove can be isolated and further prepared by recombinant DNAtechnology. As a result, the following information has been obtained,thus arriving at the present invention.

Namely, the present inventors have succeeded in cloning cDNA(complementary DNA) coding for endothelin having an amino acid sequencedifferent from those of the above endothelin-1 and endothelin-3 [humanendothelin (endothelin A) and endothelin-3] from a human cDNA library byusing as a probe the synthesized DNA segment coding for a portion ofgenomic DNA of the human endothelin described in the patent applicationspreviously filed and a DNA comprising an about 99-bp genomic DNA segmentof endothelin-2, and consequently in pioneering its mass production byrecombinant technology. The present inventors further provide aprecursor of human endothelin-2 having a novel amino acid sequence.

In accordance with the present invention, there are provided (1) a DNAcontaining a cDNA segment coding for human endothelin-2, (2) a precursorof human endothelin-2, (3) a transformant carrying a DNA containing acDNA segment coding for human endothelin-2, and (4) a method forpreparing mature human endothelin-2 which comprises culturing thetransformant described in (3), accumulating a protein in a culturemedium, and collecting the same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequences of various endothelin peptides incomparison to one another;

FIG. 2 shows a restriction enzyme fragment map of a cDNA segment codingfor human endothelin-2 obtained in the present invention;

FIGS 3-1 to 3-3 shows a nucleotide sequence (SEQ ID NO:1) of the cDNAsegment coding for human endothelin-2 obtained in the present inventionand an amino acid sequence (SEQ ID NO:2) ascertained from thatnucleotide sequence;

FIG. 4 shows an amino acid sequence of a precursor of human endothelin-2(SEQ ID NO:2);

FIG. 5 is a schematic representation showing the construction of aplasmid for expression of human endothelin in Example 5; and

FIG. 6 is a graph relating to expression of human endothelin of thepresent invention.

FIG. 7 is a reverse phase high performance chromatogram relating toexpression of human endothelin-2 of the present invention and precursorthereof.

FIG. 8 is a mass spectrogram of human big endothelin-2 (1-38).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The cDNA segment contained in the DNA of the present invention whichcodes for human endothelin-2 contains a nucleotide sequence (SEQ IDNO: 1) represented by the following formula [1] or is a portion thereof.This cDNA segment is different from the known ones and novel.

Formula [1] 1   A GGA CGC TGG CAA CAG GCA CTC CCT GCT 28 29 CCA GTC CAGCCT GCG CGC TCC ACC GCC GCT 58 59 ATG GTC TCC GTG CCT ACC ACC TGG TGCTCC 88 1  M   V   S   V   P   T   T   W   C   S 10 89 GTT GCG CTA GCCCTG CTC GTG GCC CTG CAT 118 11  V   A   L   A   L   L   V   A   L   H 20119 GAA GGG AAG GGC CAG GCT GCT GCC ACC CTG 148 21 E   G   K   G   Q   A   A   A   T   L 30 149 GAG CAG CCA GCG TCC TCATCT CAT GCC CAA 178 31  E   Q   P   A   S   S   S   H   A   Q 40 179 GGCACC CAG CTT CGG CTT CGC CGT TGC TCG 208 41 G   T   W   L   R   L   R   R   C   S 50 209 TGC AGC TCC TGG CTC GACAAG GAG TGC GTC 238 51  C   S   S   W   L   D   K   E   G   V 60 239 TACTTC TGC CAC TTG GAC ATC ATC TGG GTG 268 61 Y   F   C   H   L   D   I   I   W   V 70 269 AAC ACT CCT GAA CAG ACAGCT CCT TAC GGC 298 71  N   T   P   E   Q   T   A   P   Y   G 80 299 CTGGGA AAC CCG CCA AGA CGC CGG CGC CGC 328 81 L   G   N   P   P   R   R   R   R   R 90 329 TCC CTG CCA AGG CGC TGTCAG TGC TCC ACT 358 91  S   L   P   R   R   C   Q   C   S   S 100 359GCC AGG GAC CCC GCC TGT GCC ACC TTC TGC 388 101 A   R   D   P   A   C   A   T   F   C 110 389 CTT CGA AGG CCC TGG ACTGAA GCC GGG GCA 418 111  L   R   R   P   W   T   E   A   G   A 120 419GTC CCA AGC CGG AAG TCC CCT GCA GAC GTG 448 121 V   P   S   R   K   S   P   A   D   V 130 449 TTC CAG ACT GGC AAG ACAGGG GCC ACT ACA 478 131  F   Q   T   G   K   T   G   A   T   T 140 479GGA GAG CTT CTC CAA AGG CTG AGG GAC ATT 508 141 G   E   L   L   Q   R   L   R   D   I 150 509 TCC ACA GTC AAG AGC CTCTTT GCC AAG CGA 538 151  S   T   V   K   S   L   F   A   K   R 160 539CAA CAG GAG GCC ATG CGG GAG CCT CGG TCC 568 161 Q   Q   E   A   M   R   E   P   R   S 170 569 ACA CAT TCC AGG TGG AGGAAG AGA TAG TGT 598 171  T   H   S   R   W   R   K   R   * 179 599 CGTGAG CTG GAG GAA CAT TGG GAA GGA AGC 628 629 CCG CGG GGA GAG AGG AGG AGAGAA GTG GCC 658 659 AGG GCT TGT GGA CTC TCT GCC TGC TTC CTG 688 689 GACCGG GGC CTT GGT CCC AGA CAG CTG GAC 718 719 CCA TTT GCC AGG ATT GGC ACAAGC TCC CTG 748 749 GTG AGG GAG CCT CGT CCA AGG CAG TTC TGT 778 779 GTCCTC GCA CTG CCC AGG GAA GCC CTC GGC 808 809 CTC CAG ACT GCG GAG CAG CCTCCA GTG CTG 838 839 GCT GCT GGC CCA CAG CTC TGC TGG AAG AAC 868 869 TGCATG GGG AGT ACA TTC ATC TGG AGG CTG 898 899 CGT CCT GAG GAG TGT CCT GTCTGC TGG GCT 928 929 ACA AAC CAG GAG CAA CCG TGC AGC CAC GAA 958 959 CACGCA TGC CTC AGC CAG CCC TGG AGA CTG 988 989 GAT GGC TCC CCT GAG GCT GGCATC CTG GCT 1018 1019 GGC TGT GTC CTC TCC AGC TTT CCC TCC CCA 1048 1049GAG TTC TTG CAC CCT CAT TCC CTC GGG ACC 1078 1079 CTC CCA GTG AGA AGGGCC TGC TCT GCT TTT 1108 1109 CCT GTC TGT ATA TAA CTT ATT TGC CCT AAG1138 1139 AAC TTT GAG AAT CCC AAT TAT TTA TTT TAA 1168 1169 TGT ATT TTTTAG ACC CTC TAT TTA CCT GCG 1198 1199 AAC TTG TGT TTA TAA TAA AT 1218

The precursor of human endothelin-2 of the present invention contains anamino acid sequence (SEQ ID NO:2) represented by the following formula[2]:

          10        20        30        40        50        60 Formula[2] N-MVSVPTTWCSVALALLVALHEGKGQAAATLEQPASSSHAQGTHLRLRRCSCSSWLDKECV          70        80        90       100       110       120  YFCHLDIIWVNTPEQTAPYGLGNPPRRRRRSLPRRCQCSSARDPACATFCLRRPWTEAGA         130       140       150       160       170  VPSRKSPADVFQTGKTGATTGELLQRLRDISTVKSLFAKRQQEAMREPRSTHSRWRKR* -C

A protein having a sequence corresponding to the sequence of the 49th tothe 85th amino acids in formula 2, a sequence represented by thefollowing formula, is also a kind of precursor of human endothelin-2,and called big endothelin-2(1-37).

C S C S S W L D K E C V Y F C H L D I I W V N T P E Q T A P Y G L G N PP  (SEQ ID NO:7)

A protein having a sequence corresponding to the sequence of the 49th tothe 86th amino acids in formula 2, a sequence represented by thefollowing formula, is also a kind of precursor of human endothelin-2,and called big endothelin-2(1-38).

C S C S S W L D K E C V Y F C H L D I I W V N T P E Q T A P Y G L G N PP R  (SEQ ID NO:8)

The nucleotide sequence represented by formula [1] (SEQ ID NO:1) is thesequence of the human endothelin-2 cDNA obtained in the presentinvention. Examples of the nucleotide sequences coding for the maturehuman endothelin-2 amino acids represented by formula [2] (SEQ ID NO:2)(the above underlined sequence of C S C S S W L D K E C V Y F C H L D II W) include the sequence represented by Nos. 203 to 265 in formula [1](SEQ ID NO:1).

In the present invention, for example, an expression vector having theDNA sequence containing the nucleotide sequence coding for mature humanendothelin-2 can be prepared by the following process:

(a) Messenger RNA (mRNA) is isolated from human endothelin-2-producingcells.

(b) Single stranded complementary DNA (cDNA) is synthesized from theMRNA, followed by synthesis of double stranded DNA.

(c) The complementary DNA is introduced in a cloning vector such as aphage or a plasmid.

(d) Host cells are transformed with the recombinant phage or plasmidthus obtained.

(e) After cultivation of the transformants thus obtained, the plasmidsor the phages containing the desired DNA is isolated from thetransformant by an appropriate method such as hybridization with a DNAprobe coding for a portion of human endothelin-2 or immunoassay using ananti-human endothelin-2 antibody.

(f) The desired cloned DNA sequence is cleaved from the recombinant DNA.

(g) The cloned DNA sequence or a portion thereof is ligated downstreamfrom a promoter in the expression vector.

The mRNA coding for human endothelin-2 can be obtained from variousendothelin-producing cells such as ACHN cells of human renal cancercells.

Methods for preparing the mRNA from the human endothelin-2-producingcells include the guanidine thiocyanate method [J. M. Chirgwin et al.,Biochemistry 18, 5294 (1979)] and the like.

Using the MRNA thus obtained as a template, cDNA is synthesized by useof reverse transcriptase, for example, in accordance with the method ofH. Okayama et al. [Molecular and Cellular Bioloqy 2, 161 (1979); andibid. 3, 280 (1983)]. The cDNA thus obtained is introduced into theplasmid.

The plasmids into which the cDNA may be introduced include, for example,pBR322 [Gene 2, 95 (1977)], pBR325 [Gene 4, 121 (1978)], pUC12 [Gene 19,259 (1982)], pUC13 [Gene 19, 259 (1982)], pUC118 and pUC119, eachderived from Escherichia coli, and pUB110 derived from Bacillus subtilis[Biochemical and Biophysical Research Communication 112, 678 (1983)].However, any other plasmid can be used as long as it is replicable andgrowable in the host cell. Examples of the phage vectors into which thecDNA may be introduced include gt11 [R. Young and R. Davis, Proc. Natl.Acad. Sci. U.S.A. 80, 1194 (1983)]. However, any other phage vector canbe used as long as it is growable in the host cell.

Methods for introducing the cDNA into the plasmid include, for example,the method described in T. Maniatis et al., Molecular Cloning, ColdSpring Harbor Laboratory, p.239 (1982). Methods for introducing the cDNAinto the phage vector include, for example, the method of T. V. Hyunh etal. [DNA Cloning, A Practical Approach 1, 49 (1985)].

The plasmid thus obtained is introduced into an appropriate host cellsuch as Escherichia and Bacillus.

Examples of Escherichia described above include Escherichia coli K12DH1[Proc. Natl. Acad. Sci. U.S.A. 60, 160 (1968)], M103 [Nucleic AcidsResearch 9, 309 (1981)], JA221 [Journal of Molecular Biology 120, 517(1978)], HB101 [Journal of Molecular Biology 41, 459 (1969)] and C600[Genetics 39, 440 (1954)].

Examples of Bacillus described above include Bacillus subtilis MI114[Gene 24, 255 (1983)] and 207-21 [Journal of Biochemistry 95, 87(1984)].

Methods for transforming the host cell with the plasmid include, forexample, the calcium chloride method or the calcium chloride/rubidiumchloride method described in T. Maniatis et al., Molecular Cloning, ColdSpring harbor Laboratory, p.249 (1982).

When the phage vector is used, for example, it can be transduced intoproliferated E. coli, using the in vitro packaging method [T. Maniatiset al., Molecular Cloning, A Laboratory Manual, Coldspringharbor, 1982,pp.262-268].

Human cDNA libraries containing human endothelin-2 cDNA can be obtainedby the above-mentioned methods and the like.

Methods for cloning human endothelin-2 cDNA from the human DNA libraryinclude, for example, the plaque hybridization method using phage vectorλcharon 4A and an oligonucleotide chemically synthesized on the basis ofthe amino acid sequence of human endothelin-2 as a probe [T. Maniatis etal., Molecular Cloning, Cold Spring Harbor Laboratory, (1982)]. Thehuman endothelin-2 cDNA thus cloned may be subcloned, for example, inpBR322, pUC12, pUC13, pUC18, pUC19, pUC118 and pUC119 to obtain thehuman endothelin-2 cDNA, if necessary.

The nucleotide sequence of the DNA sequence thus obtained is determined,for example, by the Maxam-Gilbert method [A. M. Maxam and W. Gilbert,Proc. Natl. Acad. Sci. U.S.A. 74, 560 (1977)] or the dideoxy method [J.Messing et al., Nucleic Acids Research 9, 309 (1981)], and the existenceof the human endothelin-2 DNA is confirmed in comparison with the knownamino acid sequence.

As described above, the cDNA (human endothelin-2 cDNA) represented byformula [1] (SEQ ID NO:1) coding for human endothelin-2 is obtained.

FIG. 2 shows the restriction enzyme fragment map of the cDNA coding forhuman endothelin-2 obtained in Example 2 described below. FIG. 3 showsthe nucleotide sequence of the cDNA as determined by the dideoxy method(SEQ ID NO:1), and the amino acid sequence deduced from that nucleotidesequence. FIG. 4 shows the amino acid sequence of a precursor of humanendothelin-2 (SEQ ID NO:2).

The cDNA coding for human endothelin-2 cloned as described above can beused as it is, or after digestion with a restriction enzyme if desired,depending on the intended use.

A region intended to be expressed is cleaved from the cloned cDNA andligated downstream from the promoter in a vehicle (vector) suitable forexpression, whereby the expression vector can be obtained.

The cDNA has ATG as a translation initiating codon at the 5′-terminusthereof and may have TAA, TGA or TAG as a translation terminating codonat the 3′-terminus. The translation initiating codon and translationterminating codon may be added by use of an appropriate synthetic cDNAadaptor. The promoter is further ligated upstream therefrom for thepurpose of expressing the cDNA.

Examples of the vectors include the above plasmids derived from E. colisuch as pBR322, pBR325, pUC12 and pUC13, the plasmids derived fromBacillus subtilis such as pUB110, pTP5 and pC194, plasmids derived fromyeast such as pSH19 and pSH15, bacteriophages such as λphage, and animalviruses such as retroviruses and vaccinia viruses.

As the promoter used in the present invention, any promoter is availableas long as it is suitable for expression in the host cell selected forthe gene expression.

When the host cell used for transformation is Escherichia, it ispreferable that a trp promoter, a lac promoter, a recA promoter, a λPLpromoter, a 1pp promoter, etc. are used. When the host cell is Bacillus,it is preferable that a PHO5 promoter, a PGK promoter, a GAP promoter,an ADH promoter, etc. are used. In particular, it is preferable that thehost cell is Escherichia and the promoter is the trp promoter or the λPLpromoter.

When the host cell is an animal cell, a SV-40 derived promoter, aretrovirus promoter, a metallothionein promoter, a heat shock promoter,etc. are each usable.

An enhancer is also effectively used for expression.

Using a vector containing the cDNA coding for the human endothelin-2mature peptide thus constructed, transformants are prepared.

The host cells include, for example, Escherichia, Bacillus, yeast andanimal cells.

As specific examples of the above Escherichia and Bacillus, strainssimilar to those described above can be mentioned.

Examples of the above yeast include Saccharomyces cetevisiae AH22,AH22R⁻, NA87-11A and DKD-5D.

Examples of the animal cells include monkey cell COS-7, Vero, Chinesehamster cell (CHO), mouse L cell and human FL cell.

The transformation of the above Escherichia is carried out according to,for example, the method described in Proc. Natl. Acad. Sci. U.S.A. 69,2110 (1972).

The transformation of the above Bacillus is conducted according to, forexample, the method described in Molecular & General Genetics 168, 111(1979).

The transformation of the yeast is carried out according to, forexample, the method described in Proc. Natl. Acad. Sci. U.S.A. 75, 1929(1978).

The transformation of the animal cells is carried out according to, forexample, the method described in Virology 52, 456 (1973).

Thus, transformants are obtained which are transformed with anexpression vector containing the cDNA coding for the human endothelin-2mature peptide.

When bacterial transformants are cultured, a liquid medium isparticularly suitable as a medium used for culture. Carbon sources,nitrogen sources, inorganic compounds and others necessary for growth ofthe transformants are contained therein. Examples of the carbon sourcesinclude glucose, dextrin, soluble starch and sucrose. Examples of thenitrogen sources include inorganic or organic materials such as ammoniumsalts, nitrates, corn steep liquor, peptone, casein, meat extracts,soybean meal and potato extract solution. The inorganic compoundsinclude, for example, calcium chloride, sodium dihydrogenphosphate andmagnesium chloride. Yeast, vitamins, growth promoting factors and so onmay be further added thereto.

The pH of the medium is preferably about 5 to 8.

As the medium used for cultivation of Escherichia, for example, M9medium containing glucose and Casamino Acids (Miller, Journal ofExperiments in Molecular Genetics 431-433, Cold Spring HarborLaboratory, New York, 1972) is preferably used. In order to make thepromoter act efficiently, a compound such as 3-indolylacrylic acid maybe added thereto if necessary.

When the host cell is Escherichia, the cultivation is usually carriedout at about 15 to 43° C. for about 3 to 24 hours, with aeration oragitation if necessary.

When the host cell is Bacillus, the cultivation is usually carried outat about 30 to 40° C. for about 6 to 24 hours, with aeration oragitation if necessary.

When yeast transformants are cultured, for example, Burkholder minimummedium [K. L. Bostian et al., Proc. Natl. Acad. Sci. U.S.A. 77, 4505(1980)] is used as the medium. The pH of the medium is preferablyadjusted to about 5 to 8. The cultivation is usually carried out atabout 20 to 35° C. for about 24 to 72 hours, with aeration or agitationif necessary.

When animal cell transformants are cultured, examples of the mediainclude MEM medium containing about 5 to 20% fetal calf serum [Science122, 501 (1952)], DMEM medium [Virology 8, 396 (1959)], RPMI1640 medium(The Journal of the American Medical Association 199, 519 (1967)] and199 medium [Proceeding of the Society for the Biological Medicine 73, 1(1950). The pH is preferably about 6 to 8. The cultivation is usuallycarried out at about 30 to 40° C. for about 15 to 60 hours, withaeration or agitation if necessary.

The human endothelin-2 mature peptide (endothelin-2) can be isolated andpurified from the culture described above, for example, by the followingmethod.

When the human endothelin-2 mature peptide is extracted from thecultured cells, the cells are collected by methods known in the artafter cultivation. Then, the collected cells are suspended in anappropriate buffer solution and disrupted by ultrasonic treatment,lysozyme and/or freeze-thawing. Thereafter, a crude extracted solutionof the human endothelin-2 mature peptide is obtained by centrifugationor filtration. The buffer solution may contain a protein denaturant suchas urea or guanidine hydrochloride, or a surface-active agent such asTriton X-100.

When the human endothelin-2 precursor protein or mature peptide issecreted in the culture solution, a supernatant is separated from thecells by methods known in the art after the conclusion of cultivation,and then collected.

The separation and purification of the human endothelin-2 precursorprotein or mature peptide contained in the culture supernatant or theextracted solution thus obtained can be performed by an appropriatecombination of separating and purifying methods known in the art. Theknown separating and purifying methods include methods utilizingsolubility such as salt precipitation and solvent precipitation, methodsmainly utilizing a difference in molecular weight such as dialysis,ultrafiltration, gel filtration and SDS-polyacrylamide gelelectrophoresis, methods utilizing a difference in electric charge suchas ion-exchange column chromatography, methods utilizing specificaffinity such as affinity chromatography, methods utilizing a differencein hydrophobicity such as reverse phase high performance liquidchromatography and methods utilizing a difference in isoelectric pointsuch as isoelectro-focussing electrophoresis.

The activity of the human endothelin-2 precursor protein or maturepeptide thus formed can be measured by an enzyme immunoassay using aspecific antibody. If the products have vasoconstrictive activity, thehuman endothelin-2 precursor protein or mature peptide may also bemeasured based thereupon.

The cells transfected or transformed with the DNA of the presentinvention allow large amounts of the human endothelin-2 mature peptideto be produced. Hence, the production of these peptides can beadvantageously achieved.

Like the other endothelin peptides, endothelin-2 and the precursorthereof prepared here not only can be utilized as a hypotoniatherapeutic agent or a topical vasoconstrictor, but can also be utilizedto analyze the mechanism of the vasoconstrictor reactions in vivo and toelucidate the antagonists to the vasoconstrictor factors. Similarly, thepeptides have such effects as preventing various kinds of hemorrhage,for example, gastric or esophageal hemorrhage as a vasoconstrictor, andmay also be useful in curing various shock symptoms. These peptides canbe administered orally, locally, intravenously or parenterally,preferably topically or intravenously. The dose is 0.001 μg to 100μg/kg, and preferably 0.01 μg to 10 μg/kg. The peptides are used in dosedependent on weight and in the form of a solution in 1 to 10 ml ofphysiological saline.

The peptides of the present invention can be formed into variouspreparations together with additional components, such as emulsions,hydrated mixtures, tablets, solutions, powders, granules, capsules andpills. Examples of the additional components include pharmaceuticallyacceptable excipients, disintegrators, lubricants, binders, dispersants,plasticizers, fillers and carriers. As to the additional components,examples of the excipients include lactose, glucose and white sugar;those of the disintegrators include starch, sodium alginate, agar powderand carboxymethyl cellulose calcium; those of the lubricants includemagnesium stearate, talc and liquid paraffin; those of the bindersinclude syrup, gelatin solution, ethanol and polyvinyl alcohol; those ofthe dispersants include methyl cellulose, ethyl cellulose and shellac;and those of the plasticizers include glycerin and starch.

When nucleotides, amino acids and so on are indicated by abbreviationsin this specification and drawings, the abbreviations adopted by theIUPAC-IUB Commission on Biochemical Nomenclature or commonly used in theart are employed. For example, the following abbreviations are used.When the amino acids are capable of existing as optical isomers, it isunderstood that the L-forms are represented unless otherwise specified.

DNA: Deoxyribonucleic acid

cDNA: Complementary deoxyribonucleic acid

A: Adenine

T: Thymine

G: Guanine

C: Cytosine

RNA: Ribonucleic acid

mRNA: Messenger ribonucleic acid

dATP: Deoxyadenosine triphosphate

dTTP: Deoxythymidine triphosphate

dGTP: Deoxyguanosine triphosphate

dCTP: Deoxycytidine triphosphate

ATP: Adenosine triphosphate

EDTA: Ethylenediaminetetraacetic acid

SDS: Sodium dodecyl sulfate

Gly or G: Glycine

Ala or A: Alanine

Val or V: Valine

Leu or L: Leucine

Ile or I: Isoleucine

Ser or S: Serine

Thr or T: Threonine

Cys or C: Cysteine

Met or M: Methionine

Glu or E: Glutamic acid

Asp or D: Aspartic acid

Lys or K: Lysine

Arg or R: Arginine

His or H: Histidine

Phe or F: Phenylalanine

Tyr or Y: Tyrosine

Trp or W: Tryptophan

Pro or P: Proline

Asn or N: Asparagine

Gln or Q: Glutamine

With respect to human endothelin-2 of the present invention, a portionof the amino acid sequence may be modified, namely there may beaddition, elimination or substitution with other amino acids as long asthe vasoconstrictor property is not lost.

The present invention will hereinafter be described in more detail withthe following Reference Example and Examples. It is understood of coursethat these Reference Example and Examples are not intended to limit thescope of the invention.

Transformant E. coli MV1184/pHET-2(K) obtained in Example 3 wasdeposited with the Institute for Fermentation, Osaka, Japan (IFO) underthe accession number IFO 15064 on Jul. 10, 1990, and also deposited withthe Fermentation Research Institute, the Agency of Industrial Scienceand Technology, the Ministry of International Trade and Industry, Japan(FRI) under the accession number FERM BP-3008 on Jul. 12, 1990.

Transformant E. coli MC1061/P3/pTS612 obtained in Example 5 wasdeposited with the Institute for Fermentation, Osaka, Japan (IFO) underthe accession number IFO 15122 on Dec. 24, 1990, and also deposited withthe Fermentation Research Institute, the Agency of Industrial Scienceand Technology, the Ministry of International Trade and Industry, Japan(FRI) under the accession number FERM BP-3211 on Dec. 20, 1990.

Reference Example

(1) Assay of Vascular Smooth Muscle Constrictor Activity

Porcine right coronary artery spiral specimens (0.5×20 mm) with theintima denuded by rubbing with a small swab are suspended in 3 ml ofKrebs-Ringer solution maintained at 37° C. and saturated with a mixedgas containing 5% carbon dioxide and 95% oxygen by volume. After settingthe basal tension to 2 g, the isometric tension is measured with atension transducer.

(2) Assay of Cardiotonic Action

Instead of the porcine right coronary artery spiral specimens used inthe assay described in the above item (1), suspended guinea pig rightatrium specimens are used, and the tension and the heart rate per minuteare measured according to the same procedure as described in (1).

EXAMPLE 1

Preparation of DNA Probe Coding For a Portion of DNA of Genomic HumanEndothelin-2

A DNA probe coding for a portion of the DNA sequence of genomic humanendothelin-2, which had the following sequence, was chemicallysynthesized, and used for hybridization with an ACHN cell-derived cDNAlibrary.

5′CATGCCCAAGGCACCCACCTTCGGCTTCGCCGTTGCTCCTGCAGCTCCTGGCTCGACAAGGAGTGCGTCTACTTCTGCCACTTGGACATCATCTGGGTG3′  (SEQ ID NO:9)

EXAMPLE 2

Extraction of mRNA and Preparation of cDNA Library (1) Extraction ofTotal RNA From ACHN Cells

Human renal cancer cells, ACHN (ATCC, CRL 1611), were cultured inDulbecco's minimum essential medium (D-MEM) containing 10% fetal calfserum in an atmosphere of 5% CO₂ and 95% air at 37° C. [for ten 150-cm²flasks (Falcon)]. After 3 to 4 days, the culture solution was removed,followed by washing with phosphate buffered saline (PBS). Then, 100 mlper flask of a guanidine-isothiocyanate solution (5 Mguanidine-isothiocyanate, 50 mM Tris-HCl, 10 mM EDTA and 5%mercaptoethanol) was added thereto to solve the cells, followed bycollection in a 150-ml glass homogenizer and sufficient trituration.

A 20% sodium sarcosinate solution (20% sodium N-lauroylsarcosinate, 50mM Tris-HCl (pH 7.6), 10 mM EDTA and 5% mercaptoethanol) was added to afinal concentration of 5%, followed by further sufficient trituration.Solid cesium chloride was added thereto to a concentration of 0.2 g/ml,followed by further sufficient trituration while dissolving it. Then, 5ml of a 5.2 M solution of cesium chloride in 0.5 Methylenediaminetetraacetic acid (EDTA) was placed in a tube for an SW28rotor (Beckmann), and 30 ml of the above-mentioned cell solution waslayered thereon, followed by centrifugation with the SW28 rotor at28,000 rpm at 22° C. for 48 hours.

RNA was collected on pellets in the tube, and an upper layer solutionwas removed by absorption according to the method of Kaplain [Biochem.J. 183, 181-184 (1979)]. Then, after the RNA was dissolved in a 0.4%sarcosine solution, NaCl was added thereto to a final concentration of0.25 M. Cold ethanol was added thereto in an amount of 2.5 volumes, andthe resulting solution was stored at −2°.

(2) Separation of PolyA⁺ RNA

Total RNAs were collected by centrifugation (at 25,000 rpm for 20minutes), and polyA⁺ RNA was partially purified by oligo(dt) columnchromatography according to the manual of the method for purifyingpolyA⁺ RNA (Pharmacia). About 5% of the total RNAs was recovered as thepolyA⁺ RNA.

(3) Synthesis of cDNA and Preparation of cDNA Library By Using Phageλgt11

cDNA was synthesized from 5 μg of the polyA⁺ RNA obtained in (2)described above, using a cDNA synthesizing kit (Amersham) according tothe manual, and using ³²P-dCTP as a marker for cDNA synthesis. About 10%of 5 μg of the polyA⁺ RNA used was obtained as double-stranded DNA (dsDNA).

Similarly using a cDNA cloning kit (Amersham), adapters for restrictionenzyme EcoRI were ligated to this ds DNA by the ligation reaction, andtreated with EcoRI to obtain ds DNA to the 5′- and 3′-termini of whichEcoRI adapters were ligated.

The cDNA to which the EcoRI adapters were ligated was introduced intothe EcoRI site of phage λgt11 DNA, and reconstructed to phage particles,thereby obtaining a cDNA library of phage λgt11. The resulting cDNAlibrary of phage λgt11 exhibited an infection value of about 2×10⁶plaque forming units/ml.

EXAMPLE 3

Cloning of Human Endothelin-2 cDNA and Isolation of Phage Clones HavingHuman Endothelin-2 cDNA

The recombinant phage (λgt11) obtained in (3) of Example 2 wastransfected in E. coli Y1090 under a condition of 1,500 plaques/Schaleby the method of Pentone and Davis [Science 196, 180-182 (1977)], andthen the phage was transferred to a nitrocellulose filter. The filterwas treated according to the method of Benton and Davis [W. D. Bentonand R. W. Davis, Science, 196, p.180 (1977)]. A partial DNA fragmenthaving a nucleic acid sequence [Inoue et al., Proc. Natl. Acad. Sci.U.S.A. 86, 2863-2867 (1989)] coding for endothelin-2 of genomic humanDNA, which had the following sequence (SEQ ID NO:9), was synthesizedwith a DNA synthesizer (Model AB1), and a ³²P-labeled probe was preparedby the random priming method using α-³²P.dCTP.

 5′CATGCCCAAGGCACCCACCTTCGGCTTCGCCGTTGCTCCTGCAGCTCCTGGCTCGACAAGGAGTGCGTCTACTTCTGCCACTTGGACATCATCTGGGTG3′

The recombinant phage DNA transferred to the nitrocellulose filter washybridized with the ³²P-labeled endothelin-2 probe by the method ofPentone and Davis [Science 196, 180-182 (1977)]. Phage clones whichformed hybrids were screened to obtain several positive plaques. Thecloned phage was transfected in E. coli Y1090 in LB broth at 37° C. for5 to 6 hours. After removal of E. coli by centrifugation, 200 μl of a20% polyethylene glycol 0.15 M NaCl solution was added to 1 ml of theculture solution, followed by standing at 4° C. for 2 to 3 hours. Then,phage particles were precipitated by centrifugation at 15,000 rpm for 20minutes. The phage particles were suspended by addition of 100 μl ofdistilled water, and the resulting suspension was heated at 95° C. for10 minutes. This recombinant phage DNA was λgt11. Hence, using the DNAfragments of 5′ GGTGGCGACGACTCCTGGAGCCCG (SEQ ID NO:10) and 5′TTGACACCAGACCAACTGGTAATG (SEQ ID NO:11) close to the EcoRI site, arecombinant site of cDNA, as primers for λgt11, this cDNA portion wasobtained by amplification according to the polymer chain reaction (PCR)method [I. A. Michael and Iunis, PCR Protocols, Academic Press (1990)].

The DNA obtained by amplification synthesis according to the PCR methodwas treated with EcoRI, followed by ligation to the EcoRI site ofplasmid pUC118 by the ligation reaction. Then, E. coli DH5α wastransformed into 5 clones shown below:

pHET-2(52)/E. coli MV1184

pHET-2(31)/E. coli MV1184

pHET-2(27)/E. coli MV1184

pHET-2(26)/E. coli MV1184

pHET-2(35)/E. coli MV1184

Of these clones, pHET-2(52)/E. coli MV1184 is referred to aspHET/2(K)/E. coli MV1184.

EXAMPLE 4

Determination of Nucleotide Sequence of Cloned DNA

The plasmid DNA obtained in Example 3 described above was purified, andthe nucleotide sequence of the DNA was determined by the dideoxy-chaintermination method [Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467 (1977)].The determination direction and the whole nucleotide sequence (SEQ IDNO:1) thereof are shown in FIG. 3.

The cDNA coding for the precursor of human endothelin-2 is pHET-2(52)and consists of 1218 nucleotides. An open frame of 534 nucleotides isremoved therefrom to code for 178 amino acid residues. The portion beingboxed is the portion of human endothelin-2, and identical with thesequence previously shown from the sequence of the genomic DNA.

EXAMPLE 5

Expression of Human Endothelin-2

Plasmid pHET-2(K) having cDNA coding for the precursor of humanendothelin-2 was cleaved with the restriction enzyme EcoRI andintroduced into the EcoRI site of expression vector pcDNA. Then, E. coliMC1061/P3 was transformed to obtain E. coli MC1061/P3/pTS612 (FIG. 5).This plasmid was purified and allowed to be entrapped in CHO-K1 cellstogether with plasmid pSV2neo according to the method of McCutchan andPagano [J. H. McCutchan and J. S. Pagano, J. of the National CancerInstitute, 41(2), pp. 351-357 (1968)]. Then, drug G418-resistant cellswere selected. The cells were cloned and the cell line was named asCHO-12-5-12.

The selected cells were cultured in DMEM medium containing 10% calfserum, and the immunoreactive ET of the culture solution was assayed. Asa result, secretion of immunoreactive endothelin (closed squares) andbig-endothelin (open squares) was observed, in addition to cellproliferation (open triangles show the total number of cells), as shownin FIG. 6.

EXAMPLE 6

Expression of Human Endothelin and Endothelin Precursor in CHO CellTransformant and Determination of the Structures Thereof

1,500 ml of a culture supernatant of CHO-12-5-12 cells, CHO cellstransformed with expression plasmid pTS612 into which the cDNA of humanendothelin-2 was introduced, was divided into three portions, and each500 ml portion was treated in the following manner.

(1) Each portion was filtered through a 0.22-μm filter, and (2)subjected to AwETN40-affinity column chromatography (the way to prepareis described below). (3) The absorbed substances were separated byreverse phase high performance liquid chromatography (RP-HPLC) (FIG. 7).(4) Each of the resulting fractions was dried under vacuum, and (5)subjected to the enzyme immunoassay (EIA) of human big-endothelin-2(1-37). (6) A fraction (peak 24 in FIG. 7) eluted 2 minutes beforeelution of synthesized human big-endothelin-2 (1-37) was taken, and (7)the amino acid sequence was determined with an AB1 (model 477A).

As a result, the sequence of 20 amino acid residues from the N-terminuswas determined asXaa-Ser-Xaa-Ser-Ser-Xaa-Leu-Asp-Lys-Glu-Xaa-Val-Tyr-Phe-Xaa-(His)-Leu-Asp-Ile-Ile-Xaa(SEQ ID NO:12).

The seventh Leu from the N-terminus apparently corresponded to Leu atthe time that the nucleotide sequence of the cDNA of endothelin-2 wastranslated into the amino acid sequence. The N-terminus was deduced asCys, and the above sequence was deduced as the sequence of endothelin-2,considering positions 1, 3, 11 and 15 of Cys.

Although the N-terminal amino acid sequence of immunoreactive(ir)-big-endothelin-2 corresponding to peak 24 in FIG. 7 was shownabove, the fraction of peak 24 was further analyzed with massspectrographs (JMS-HX110HF and DA-500, JEOL) to determine the totalmolecular weight and the C-terminal amino acid sequence. As a result,the molecular weight was measured to be 4,342 (FIG. 8), and thefollowing sequence consisting of 38 amino acid residues in which one Argbinds to the C-terminus of amino acids 1-37 based on the nucleotidesequence of the cDNA of human big-endothelin-2 (1-37) was deduced:

(SEQ ID NO:8)   1 Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val TyrPhe Cys His Leu Asp Ile Ile Trp Val Asn Thr Pro Glu Gln Thr Ala Pro TyrGly Leu Gly Asn Pro      38 Pro Arg

This novel peptide is named human big-endothelin-2 (1-38). The moleculeeluted at the same position as this human big-endothelin-2 (1-38) ispresumed to exist also in human blood and a culture supernatant of humanrenal cancer cell ACHN, and this molecule as well as big-endothelin-2(1-37) is a precursor of endothelin-2.

The antibody column using AwETN40 was produced by the following method.

AwETN40 antibodies were allowed to bind to a tresyl TOYOPAL (TOYO ROSHILtd. Japan) column according to methods known in the art. 500 ml of theculture supernatant of CHO-12-5-12 cells was treated with 1 ml of theobtained gel. Namely, a 1 cm diameter column was packed with theantibody-binding column to height of 0.5 cm, and a sample for RP-HPLCwas prepared by the following steps:

(1) 8 ml of 0.05 M glycine-HCl and 0.1 M NaCl was allowed to flow.

(2) 30 ml of phosphate buffer was allowed to flow.

(3) 500 ml of the cell culture supernatant was allowed to flow.

(4) T he column was washed with 30 ml of phosphate buffer.

(5) The column was washed with 10 ml of distilled water.

(6) Elution was conducted with 9 ml of 60% CH₃CN and 0.1% TFA.

Then, the eluate was concentrated to 200 to 300 μl in the presence of N₂gas, and subjected to RP-HPLC to obtain immunoreaction-positivesubstances, endothelin-2 and big-endothelin-2, by fractionation (FIG.7).

The conditions of RP-HPLC were as follows:

Column: TSKgel ODS-80 (TOSO Ltd. Japan, 4.6 mm 1D×25 cm)

Eluent A: 5% CH₃CN, 0.05% TFA

Eluent B: 60% CH₃CN, 0.05% TFA

Elution was effected by a linear gradient of CH₃CN from 18 to 60%. Aportion of each fraction was taken and assayed by the EIA of human bigendothelin-2 (1-37).

Thus, the endothelin precurs or containing 21 amino acid residues ofendothelin-2 (ET-2) was expressed in the CHO-K1 cell, an animal cell,and endothelin-2, big-endothelin-2 (big-ET-2)(1-37) and big-ET-2(1-38)larger in molecule than ET-2 and proteins having larger molecules wereallowed to be secreted in the cell culture solution.

This system is useful to know the synthesis and secretion mechanisms ofET-2, and to develop methods for inhibiting the synthesis and secretionthereof. Further, this system is available for developing agents forinhibiting the synthesis of three kinds of endothelin peptides includingpeptides related to endothelin-l and endothelin-3.

As described above, the cDNA coding for the precursor of humanendothelin-2 was cloned, and the structure thereof was determined. ThiscDNA was introduced into the expression vector, whereby it becamepossible to express endothelin-2 and the precursor thereof.

Further, using the cDNA of human endothelin-2 as the probe, it becamepossible to elucidate the gene expression of endothelin-2 at the DNAlevel, along with endothelin-1 and endothelin-3 hitherto obtained by thepresent inventors. Furthermore, it became possible to diagnose diseasesto which endothelin was related, in combination with the EIA ofendothelin previously developed.

The following references, which are referred to for their disclosures atvarious points in this application, are incoporated herein by reference.

Proc. Natl. Acad. Sci. U.S.A. 79, 5485 (1982)

Proc. Natl. Acad. Sci. U.S.A. 81, 4577 (1984)

Amer. J. Physiol. 248, c550 (1985)

J. Cell Physiol. 132, 263 (1987)

J. Pharmacl. Exp. Ther. 236, 339 (1985)

Circulation Research 60, 422 (1987)

Japanese Patent Application No. 255381/1987

Japanese Patent Application No. 275613/1987

Japanese Patent Application No. 313155/1987

Japanese Patent Application No. 148158/1988

Japanese Patent Application No. 174935/1988

Japanese Patent Application No. 188083/1988

Japanese Patent Application No. 278497/1989

Japanese Patent Application No. 223389/1988

Japanese Patent Application No. 274990/1989

Proc. Natl. Acad. Sci. U.S.A. 86, 2863-2867 (1989)

12 1218 base pairs nucleic acid double linear cDNA to mRNA CDS 59..592 1AGGACGCTGG CAACAGGCAC TCCCTGCTCC AGTCCAGCCT GCGCGCTCCA CCGCCGCT 58 ATGGTC TCC GTG CCT ACC ACC TGG TGC TCC GTT GCG CTA GCC CTG CTC 106 Met ValSer Val Pro Thr Thr Trp Cys Ser Val Ala Leu Ala Leu Leu 1 5 10 15 GTGGCC CTG CAT GAA GGG AAG GGC CAG GCT GCT GCC ACC CTG GAG CAG 154 Val AlaLeu His Glu Gly Lys Gly Gln Ala Ala Ala Thr Leu Glu Gln 20 25 30 CCA GCGTCC TCA TCT CAT GCC CAA GGC ACC CAC CTT CGG CTT CGC CGT 202 Pro Ala SerSer Ser His Ala Gln Gly Thr His Leu Arg Leu Arg Arg 35 40 45 TGC TCC TGCAGC TCC TGG CTC GAC AAG GAG TGC GTC TAC TTC TGC CAC 250 Cys Ser Cys SerSer Trp Leu Asp Lys Glu Cys Val Tyr Phe Cys His 50 55 60 TTG GAC ATC ATCTGG GTG AAC ACT CCT GAA CAG ACA GCT CCT TAC GGC 298 Leu Asp Ile Ile TrpVal Asn Thr Pro Glu Gln Thr Ala Pro Tyr Gly 65 70 75 80 CTG GGA AAC CCGCCA AGA CGC CGG CGC CGC TCC CTG CCA AGG CGC TGT 346 Leu Gly Asn Pro ProArg Arg Arg Arg Arg Ser Leu Pro Arg Arg Cys 85 90 95 CAG TGC TCC AGT GCCAGG GAC CCC GCC TGT GCC ACC TTC TGC CTT CGA 394 Gln Cys Ser Ser Ala ArgAsp Pro Ala Cys Ala Thr Phe Cys Leu Arg 100 105 110 AGG CCC TGG ACT GAAGCC GGG GCA GTC CCA AGC CGG AAG TCC CCT GCA 442 Arg Pro Trp Thr Glu AlaGly Ala Val Pro Ser Arg Lys Ser Pro Ala 115 120 125 GAC GTG TTC CAG ACTGGC AAG ACA GGG GCC ACT ACA GGA GAG CTT CTC 490 Asp Val Phe Gln Thr GlyLys Thr Gly Ala Thr Thr Gly Glu Leu Leu 130 135 140 CAA AGG CTG AGG GACATT TCC ACA GTC AAG AGC CTC TTT GCC AAG CGA 538 Gln Arg Leu Arg Asp IleSer Thr Val Lys Ser Leu Phe Ala Lys Arg 145 150 155 160 CAA CAG GAG GCCATG CGG GAG CCT CGG TCC ACA CAT TCC AGG TGG AGG 586 Gln Gln Glu Ala MetArg Glu Pro Arg Ser Thr His Ser Arg Trp Arg 165 170 175 AAG AGATAGTGTCGTG AGCTGGAGGA ACATTGGGAA GGAAGCCCGC GGGGAGAGAG 642 Lys ArgGAGGAGAGAA GTGGCCAGGG CTTGTGGACT CTCTGCCTGC TTCCTGGACC GGGGCCTTGG 702TCCCAGACAG CTGGACCCAT TTGCCAGGAT TGGCACAAGC TCCCTGGTGA GGGAGCCTCG 762TCCAAGGCAG TTCTGTGTCC TCGCACTGCC CAGGGAAGCC CTCGGCCTCC AGACTGCGGA 822GCAGCCTCCA GTGCTGGCTG CTGGCCCACA GCTCTGCTGG AAGAACTGCA TGGGGAGTAC 882ATTCATCTGG AGGCTGCGTC CTGAGGAGTG TCCTGTCTGC TGGGCTACAA ACCAGGAGCA 942ACCGTGCAGC CACGAACACG CATGCCTCAG CCAGCCCTGG AGACTGGATG GCTCCCCTGA 1002GGCTGGCATC CTGGCTGGCT GTGTCCTCTC CAGCTTTCCC TCCCCAGAGT TCTTGCACCC 1062TCATTCCCTC GGGACCCTCC CAGTGAGAAG GGCCTGCTCT GCTTTTCCTG TCTGTATATA 1122ACTTATTTGC CCTAAGAACT TTGAGAATCC CAATTATTTA TTTTAATGTA TTTTTTAGAC 1182CCTCTATTTA CCTGCGAACT TGTGTTTATA ATAAAT 1218 178 amino acids amino acidlinear protein 2 Met Val Ser Val Pro Thr Thr Trp Cys Ser Val Ala Leu AlaLeu Leu 1 5 10 15 Val Ala Leu His Glu Gly Lys Gly Gln Ala Ala Ala ThrLeu Glu Gln 20 25 30 Pro Ala Ser Ser Ser His Ala Gln Gly Thr His Leu ArgLeu Arg Arg 35 40 45 Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu Cys Val TyrPhe Cys His 50 55 60 Leu Asp Ile Ile Trp Val Asn Thr Pro Glu Gln Thr AlaPro Tyr Gly 65 70 75 80 Leu Gly Asn Pro Pro Arg Arg Arg Arg Arg Ser LeuPro Arg Arg Cys 85 90 95 Gln Cys Ser Ser Ala Arg Asp Pro Ala Cys Ala ThrPhe Cys Leu Arg 100 105 110 Arg Pro Trp Thr Glu Ala Gly Ala Val Pro SerArg Lys Ser Pro Ala 115 120 125 Asp Val Phe Gln Thr Gly Lys Thr Gly AlaThr Thr Gly Glu Leu Leu 130 135 140 Gln Arg Leu Arg Asp Ile Ser Thr ValLys Ser Leu Phe Ala Lys Arg 145 150 155 160 Gln Gln Glu Ala Met Arg GluPro Arg Ser Thr His Ser Arg Trp Arg 165 170 175 Lys Arg 21 amino acidsamino acid linear peptide 3 Cys Ser Cys Ser Ser Leu Met Asp Lys Glu CysVal Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp 20 21 amino acidsamino acid linear peptide 4 Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu CysVal Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp 20 21 amino acidsamino acid linear peptide 5 Cys Ser Cys Asn Ser Trp Leu Asp Lys Glu CysVal Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp 20 21 amino acidsamino acid linear peptide 6 Cys Thr Cys Phe Thr Tyr Lys Asp Lys Glu CysVal Tyr Tyr Cys His 1 5 10 15 Leu Asp Ile Ile Trp 20 37 amino acidsamino acid linear peptide 7 Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu CysVal Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp Val Asn Thr Pro GluGln Thr Ala Pro Tyr Gly 20 25 30 Leu Gly Asn Pro Pro 35 38 amino acidsamino acid linear peptide 8 Cys Ser Cys Ser Ser Trp Leu Asp Lys Glu CysVal Tyr Phe Cys His 1 5 10 15 Leu Asp Ile Ile Trp Val Asn Thr Pro GluGln Thr Ala Pro Tyr Gly 20 25 30 Leu Gly Asn Pro Pro Arg 35 99 basepairs nucleic acid single linear DNA (genomic) 9 CATGCCCAAG GCACCCACCTTCGGCTTCGC CGTTGCTCCT GCAGCTCCTG GCTCGACAAG 60 GAGTGCGTCT ACTTCTGCCACTTGGACATC ATCTGGGTG 99 24 base pairs nucleic acid single linear 10GGTGGCGACG ACTCCTGGAG CCCG 24 24 base pairs nucleic acid single linear11 TTGACACCAG ACCAACTGGT AATG 24 21 amino acids amino acid linearpeptide 12 Xaa Ser Xaa Ser Ser Xaa Leu Asp Lys Glu Xaa Val Tyr Phe XaaHis 1 5 10 15 Leu Asp Ile Ile Xaa 20

What is claimed is:
 1. An isolated and purified precursor protein havingan amino acid sequence as defined in the Sequence Listing by SEQ IDNO:2.
 2. An isolated and purified DNA coding for human endothelin-2having a nucleotide sequence represented by the nucleotide sequence ofNos. 59 to 592 of SEQ ID NO:1.
 3. A transformant carrying a DNAcontaining a cDNA segment coding for human endothelin-2 in which saidcDNA segment coding for human endothelin-2 contains a nucleotidesequence as defined in the Sequence Listing by SEQ ID NO:1.
 4. Atransformant which has the characteristics of Escherichia coliMV1183/pHET-2(K) (FERM-BP-3008).
 5. A transformant which has thecharacteristics of Escherichia coli MC 1061/p3/pTS612 (FERM-BP-3211). 6.A method for preparing a mature human endothelin-2 protein whichcomprises culturing the transformant claimed in claim 3, accumulatingthe mature endothelin-2 protein in a culture medium, and collecting thesame.